High production strains used for fermentation industry are obtained by repeated sorting and breeding improvements over several decades together with mutation processes in order to obtain a good strain having special genetic variations. Thus, these high producing mutants are undeniably the lifeline of companies and regarded as the intensive products of very important techniques. However, strain improvement by mutation process has defects such as a lot of labor and time being required, poor reproducibility, and low probability of obtaining good strains. Therefore, strain improvement has recently been advanced increasingly with gene manipulation technology as a theoretical reproducible technology.
Processes for improving the productivity of an objective substance include the increase of copy number per cell of a gene relating to the biosynthesis of the substance for enhancing its expression amount. Biosynthesis of secondary metabolites such as antibiotics requires many genes, which form a cluster on chromosome having a length extending to several ten kb. In this case, the development of technology for increasing the gene copy number of the whole cluster will produce a lot of achievements. A process for increasing the gene copy number relating to the biosynthesis of an objective substance includes cloning in a plasmid which may retain a high copy number, but high-copy type plasmids has a defect of maintaining stability, which makes it difficult to clone DNA of a long region. In addition, cosmid vector and BAC vector which have been developed for the purpose of cloning the long region DNA are currently in limited copy number for improving the stability.
It has been described in U.S. Pat. No. 5,240,858 that a certain gene region can be amplified in tandem on chromosome in Streptomyces achromogenes. However, this technology is described only as a technique which can amplify the DNA region whose size is 10 kb or less and could not be applied to the tandem amplification of giant size gene regions on a genome.
On the other hand, it has been described that a kanamycin biosynthetic gene cluster has been first cloned on 2002 (Japanese Patent Laid-Open Publication No. 2004-173537). It has been further described in the gene analysis of kanamycin high production strains used in the fermentation industry that the copy number of the kanamycin biosynthetic gene cluster has been increased (Yanai, K. & Murakami, T., Journal of Antibiotics, (Japan), 2004, Vol. 57, p. 351-354). It has been then revealed that the amplification unit containing a kanamycin biosynthetic gene cluster has a size of 145 kb in a kanamycin high production strain, and the amplification unit has been amplified to 36 or more copies (Yanai, K. et al., Proceedings of the National Academy of Sciences of the United States of America, (USA), 2006, Vol. 103, p. 9661-9666). However, the high production strain exhibiting these phenomena is the one obtained as a result of mutation processes over a long period of time and repeated sortings in order to improving productivity of kanamycin (Yanai, K. et al., Proceedings of the National Academy of Sciences of the United States of America, (USA), 2006, Vol. 103, p. 9661-9666). Thus, it has been believed impossible to reproduce the amplification phenomena in a giant size DNA region found out in a kanamycin high production strain and to find a key gene relating to it.
On the basis of the background described above, there still exists a need for a process for tandemly amplifying a giant size DNA region on a genome which may be applicable to a gene cluster required for the biosynthesis of secondary metabolites such as antibiotics.